This project aims to understand the mechanisms underlying key immune defects occurring during chronic virus infection and the ability ofthe relevant cell populations to recover after effective virologic cure. The focus of this study is Hepatitis C Virus (HCV) a pathogen infecting 170 million people globally and a major cause of liver disease and liver cancer. HCV is highly suitable for analysis of human infection because of the robust outcomes of persistent vs naturally cleared infection, and the demonstrated role of T cell subsets in this outcome.The project harnesses advances in 3 areas to allow for experimental in vivo and ex vivo study of these responses. A) The development of effective directly acting antiviral agents (DAAs) such that DAA-mediated cure may be achieved without interferon. B) The ability to induce robust antiviral T cell populations in vivo using combinations of virally vectored vaccine constructs. This protocol - using material already available - allows for controlled analysis of CD4+ and CD8+ T cell responses to vaccine and non-vaccine HCV antigens, antigens within the vector (adenovirus), and unrelated antigens. C) The definition of novel, dominant intrahepatic CD8+ T cell populations during the last U19 period - notably the CDl61++ subset, which represent up to 50% of the CD8+ T cell liver infiltrate and are dysregulated during chronic infection. The relevant populations will be studied using mulfiparametric flow cytometry and Cytof, analysis of gene expression and regulation after tetramer-sorting, and analysis of TCR usage to explore their function, phenotype and regulation. Two major aims will be addressed: AIM 1: To test whether HCV-specific memory CD4+ and CD8+ T cell pools induced in the presence or absence of HCV infection share common pathways of regulation and differentiation and assess the impact of DAA-mediated cure. AIM 2: To test whether common molecular and cellular mechanisms exist for negative regulation of distinct HCV-specific and non HCV-specific T cells, including liver-homing T cell populations and whether DAA-mediated cure can reverse this regulation.